PROJECT SUMMARY - ABSTRACT The islet lesion of human type 2 diabetes includes decreases in both ss-cell function and mass, which together contribute to the development and progression of hyperglycemia over time. Islet amyloid deposition is one factor that contributes to this loss of ss-cells and is observed in most patients with type 2 diabetes. These amyloid deposits contain as their unique peptide component islet amyloid polypeptide (IAPP), also known as amylin, which is a normal ss-cell secretory product of the ss-cell. During the process of islet amyloid formation, IAPP aggregates and is cytotoxic. This aggregation process results in the formation of fibrils and ultimately amyloid deposits that are typically located extracellularly. The development and accumulation of these deposits outside the ss-cell suggests that mechanisms controlling the normal clearance of IAPP may be impaired under conditions of amyloid formation. While we know much about islet amyloid and its toxicity, a great deal less is known about the normal mechanism(s) that decrease fibril formation by IAPP and/or enhance fibril clearance. Our recently developed data demonstrate that two proteases, neprilysin and matrix metalloproteinase-9 (MMP-9), normally produced by the islet, can limit fibril formation by IAPP. Others have shown that another islet protease, insulin-degrading enzyme (IDE), acts similarly. Based on these findings, we now propose to determine in vitro the mechanisms by which these three proteases decrease islet amyloid formation and in vivo whether neprilysin and MMP-9 are effective in limiting amyloid deposition and ss-cell toxicity. In order to achieve this, we have identified the following three major specific objectives: 1. To determine the mechanism(s) by which neprilysin decreases islet amyloid formation;2. To determine the mechanism(s) by which MMP-9 decreases islet amyloid formation;and 3. To determine the mechanism(s) by which IDE decreases islet amyloid formation. Using in vitro approaches, we will determine whether neprilysin can dissociate or degrade IAPP fibrils and/or islet amyloid deposits and whether the products of IAPP cleavage by MMP-9 and IDE are amyloidogenic. Additional in vitro studies will be done in cultured human and human IAPP (hIAPP) transgenic mouse islets using chemical inhibitors, siRNA and lentiviral constructs. In these studies we will examine whether decreasing protease activity increases amyloid deposition and ss-cell apoptosis, and whether increasing protease activity decreases amyloid deposition and ss-cell apoptosis. The in vivo studies will employ our hIAPP transgenic mouse islet transplant model of amyloid formation to determine whether increased neprilysin and MMP-9 activity produced by transplanting lentivirus-transduced islets is associated with reduced amyloid formation and decreased ss-cell loss. In summary, the goal of these studies is to enhance our knowledge of the physiology and pathophysiology of neprilysin, MMP-9 and IDE as they relate to hIAPP and its aggregated forms. By examining their role in modifying islet amyloid formation, we hope to find therapeutic targets that could be used to reduce islet amyloid and thereby preserve ss-cell mass in type 2 diabetes. PUBLIC HEALTH RELEVANCE: NARRATIVE Type 2 diabetes is reaching epidemic proportions and is common amongst Veterans. Part of the process that results in the increase in glucose levels that characterizes diabetes is the loss of pancreatic islet ss-cells that are responsible for producing and releasing insulin. This destruction of ss-cells occurs in part by the formation of amyloid deposits, which are comprised of a protein called islet amyloid polypeptide. The reasons why these deposits form are unclear. We now propose to use islets from humans and animal models to study the mechanisms responsible for the formation of these destructive amyloid deposits. The goal is to develop approaches that can be used to prevent the development of islet amyloid and can ultimately be applied to treat patients with type 2 diabetes in order to preserve their ss-cells.